RESUMEN
The ß-cell-enriched MAFA transcription factor plays a central role in regulating glucose-stimulated insulin secretion while also demonstrating oncogenic transformation potential in vitro. No disease-causing MAFA variants have been previously described. We investigated a large pedigree with autosomal dominant inheritance of diabetes mellitus or insulinomatosis, an adult-onset condition of recurrent hyperinsulinemic hypoglycemia caused by multiple insulin-secreting neuroendocrine tumors of the pancreas. Using exome sequencing, we identified a missense MAFA mutation (p.Ser64Phe, c.191C>T) segregating with both phenotypes of insulinomatosis and diabetes. This mutation was also found in a second unrelated family with the same clinical phenotype, while no germline or somatic MAFA mutations were identified in nine patients with sporadic insulinomatosis. In the two families, insulinomatosis presented more frequently in females (eight females/two males) and diabetes more often in males (12 males/four females). Four patients from the index family, including two homozygotes, had a history of congenital cataract and/or glaucoma. The p.Ser64Phe mutation was found to impair phosphorylation within the transactivation domain of MAFA and profoundly increased MAFA protein stability under both high and low glucose concentrations in ß-cell lines. In addition, the transactivation potential of p.Ser64Phe MAFA in ß-cell lines was enhanced compared with wild-type MAFA. In summary, the p.Ser64Phe missense MAFA mutation leads to familial insulinomatosis or diabetes by impacting MAFA protein stability and transactivation ability. The human phenotypes associated with the p.Ser64Phe MAFA missense mutation reflect both the oncogenic capacity of MAFA and its key role in islet ß-cell activity.
Asunto(s)
Diabetes Mellitus/genética , Hiperinsulinismo/genética , Insulinoma/genética , Factores de Transcripción Maf de Gran Tamaño/genética , Proteínas Mutantes/genética , Mutación Missense , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Femenino , Genes Dominantes , Humanos , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patología , Insulinoma/metabolismo , Insulinoma/patología , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Masculino , Proteínas Mutantes/metabolismo , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Linaje , Estabilidad Proteica , Activación Transcripcional , Secuenciación del ExomaRESUMEN
The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5' conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (-2139 to -1958â bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature ß cells in vivo Pdx1ΔAREAII/- mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon+ α relative to insulin+ ß cells, associated with the transcriptional and epigenetic derepression of the α-cell-determining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and ß cells that were incapable of maturation. Creating the Pdx1ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet ß cells carrying abnormal chromatin marking in vital ß-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and ß-cell-specific Pdx1 functions.
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Diferenciación Celular/genética , Elementos de Facilitación Genéticos , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/embriología , Transactivadores/metabolismo , Animales , Sitios de Unión/genética , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Células Secretoras de Insulina/citología , Islotes Pancreáticos/crecimiento & desarrollo , Islotes Pancreáticos/metabolismo , Mamíferos/embriología , Mamíferos/genética , Ratones , Ratones Transgénicos , Organogénesis/genética , Especificidad de la EspecieRESUMEN
AIMS/HYPOTHESIS: The molecular response and function of pancreatic islet cells during metabolic stress is a complex process. The anatomical location and small size of pancreatic islets coupled with current methodological limitations have prevented the achievement of a complete, coherent picture of the role that lipids and proteins play in cellular processes under normal conditions and in diseased states. Herein, we describe the development of untargeted tissue imaging mass spectrometry (IMS) technologies for the study of in situ protein and, more specifically, lipid distributions in murine and human pancreases. METHODS: We developed matrix-assisted laser desorption/ionisation (MALDI) IMS protocols to study metabolite, lipid and protein distributions in mouse (wild-type and ob/ob mouse models) and human pancreases. IMS allows for the facile discrimination of chemically similar lipid and metabolite isoforms that cannot be distinguished using standard immunohistochemical techniques. Co-registration of MS images with immunofluorescence images acquired from serial tissue sections allowed accurate cross-registration of cell types. By acquiring immunofluorescence images first, this serial section approach guides targeted high spatial resolution IMS analyses (down to 15 µm) of regions of interest and leads to reduced time requirements for data acquisition. RESULTS: MALDI IMS enabled the molecular identification of specific phospholipid and glycolipid isoforms in pancreatic islets with intra-islet spatial resolution. This technology shows that subtle differences in the chemical structure of phospholipids can dramatically affect their distribution patterns and, presumably, cellular function within the islet and exocrine compartments of the pancreas (e.g. 18:1 vs 18:2 fatty acyl groups in phosphatidylcholine lipids). We also observed the localisation of specific GM3 ganglioside lipids [GM3(d34:1), GM3(d36:1), GM3(d38:1) and GM3(d40:1)] within murine islet cells that were correlated with a higher level of GM3 synthase as verified by immunostaining. However, in human pancreas, GM3 gangliosides were equally distributed in both the endocrine and exocrine tissue, with only one GM3 isoform showing islet-specific localisation. CONCLUSIONS/INTERPRETATION: The development of more complete molecular profiles of pancreatic tissue will provide important insight into the molecular state of the pancreas during islet development, normal function, and diseased states. For example, this study demonstrates that these results can provide novel insight into the potential signalling mechanisms involving phospholipids and glycolipids that would be difficult to detect by targeted methods, and can help raise new hypotheses about the types of physiological control exerted on endocrine hormone-producing cells in islets. Importantly, the in situ measurements afforded by IMS do not require a priori knowledge of molecules of interest and are not susceptible to the limitations of immunohistochemistry, providing the opportunity for novel biomarker discovery. Notably, the presence of multiple GM3 isoforms in mouse islets and the differential localisation of lipids in human tissue underscore the important role these molecules play in regulating insulin modulation and suggest species, organ, and cell specificity. This approach demonstrates the importance of both high spatial resolution and high molecular specificity to accurately survey the molecular composition of complex, multi-functional tissues such as the pancreas.
Asunto(s)
Islotes Pancreáticos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Técnica del Anticuerpo Fluorescente , Gangliósidos/análisis , Humanos , Inmunohistoquímica , Ratones , PáncreasRESUMEN
PURPOSE: Sentinel lymph node biopsy (SLNB) alone has thus become an accepted surgical approach for patients with limited axillary metastatic disease. We investigated to what extent isolated tumor cells (ITC) or micrometastasis in SLNBs is associated with proven tumor cells or metastasis in non-sentinel lymph nodes. Furthermore, we investigated the feasibility of SLNB in multifocal and multicentric tumors as both entities have been considered a contraindication for this technique. METHODS: 1214 women suffering from T1 and T2 invasive breast cancer, with clinically and sonographically insuspect axillary status and undergoing primary breast cancer surgery including SLNB and axillary staging in case of SLN (sentinel lymph node) metastases, were recruited into this multicentered study. RESULTS: ITC and micrometastases were found in 2.01 and 21.4% of patients with SLN metastases (n = 299). Among patients with sentinel micrometastases, 4.7% showed further axillary micrometastases, while only two patients (3.1%) had two axillary macrometastases. Multifocal and multicentric tumors were diagnosed in 9.3 and 2.6% of our patients who at least had one SLN resected, respectively. Detection rates of SLNs did not differ between the cohorts suffering from unicentric and multifocal or multicentric disease. Moreover, the portion of tumor-free SLNs, the number of SLNs with metastasis as well as the mean number of resected SLNs did not differ. CONCLUSIONS: No patient with sentinel node micrometastases showed more than two axillary macrometastases. Multifocal and multicentric disease is no contraindication for SLNB.
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Neoplasias de la Mama/epidemiología , Ganglios Linfáticos/patología , Micrometástasis de Neoplasia/patología , Biopsia del Ganglio Linfático Centinela/métodos , Ganglio Linfático Centinela/patología , Adulto , Anciano , Axila/patología , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Femenino , Humanos , Escisión del Ganglio Linfático , Metástasis Linfática , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios RetrospectivosRESUMEN
OBJECTIVE: To identify subgroups of patients with pT1 pN0 breast cancer (BC) who might not profit from adjuvant systemic therapy (AST). METHODS: Data of 3,774 pT1 pN0 BC patients from 17 certified BC centres within the BRENDA study group were collected between 1992 and 2008 and retrospectively analysed. Uni- and multivariate analyses were performed using Kaplan-Meier methods and Cox regression models. RESULTS: 279 (7.4%) of the pT1 pN0 BC patients were T1a, 944 (25.0%) were T1b and 2,551 (67.6%) were T1c. There was no significant difference (p > 0.1) in recurrence-free survival (RFS)/overall survival (OAS) between patients with pT1a, pT1b, and T1c. Patients receiving any type of AST had a better outcome compared to women without AST after adjusting for age, tumour size, and intrinsic subtypes (RFS: p < 0.001; OAS: p < 0.001). AST was the most important prognostic parameter for RFS followed by intrinsic subtypes and age. CONCLUSION: Patients with pT1 pN0 BC profit from AST independently of molecular subtypes, tumour size, age or comorbidity, with 5-year RFS of more than 95%. The correct definition of subgroups of patients who do not need AST is still an open question.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Quimioterapia Adyuvante , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Quimioterapia Adyuvante/efectos adversos , Esquema de Medicación , Femenino , Alemania/epidemiología , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Guías de Práctica Clínica como Asunto , Pronóstico , Modelos de Riesgos Proporcionales , Receptor ErbB-2 , Estudios Retrospectivos , Medición de Riesgo , Adulto JovenRESUMEN
PURPOSE: The development of metastases is the most aggressive attribute of breast cancer. In this retrospective multicenter study, we evaluated if and how the different pathological breast cancer subtypes influence the spreading of tumor cells, the development of metastasis and the survival of breast cancer patients. METHODS: This retrospective German multicenter study is based on the BRENDA collective including 9625 breast cancer patients treated in the adjuvant setting. We used the χ 2 tests for the analysis of the categorical variables between groups of patients with different sites of metastasis. Survival distributions and median survival times were estimated using the Kaplan-Meier product-limit method. The log-rank test was applied to compare survival rates. The Cox proportional hazards model was used to estimate the hazard ratio and confidence intervals. RESULTS: 886 women developed metastases during a time interval of 53 months after primary diagnosis. Luminal A tumor patients were more likely to get bone metastases than lung, liver or CNS metastases. Patients with a triple-negative subtype were, however, the least affected by metastasis in the skeleton. They were most likely to develop visceral metastases. Location, numbers of metastases herein and the subtype influenced the overall survival (OAS). Altogether, the best OAS was found in patients with the luminal A subtype, the worst in patients with the triple-negative subtype. CONCLUSIONS: Knowledge of the typical metastatic pattern of the subtypes of breast cancer will help to personalize therapeutic options and follow-up examinations of cancer patients.
Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Femenino , Humanos , Estimación de Kaplan-Meier , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
The murine Mafa transcription factor is a key regulator of postnatal islet ß-cell activity, affecting insulin transcription, insulin secretion, and ß-cell mass. Human MAFA expression is also markedly decreased in islet ß-cells of type 2 diabetes mellitus (T2DM) patients. Moreover, levels are profoundly reduced in db/db islet ß-cells, a mouse model of T2DM. To examine the significance of this key islet ß-cell-enriched protein to glycemic control under diabetic conditions, we generated transgenic mice that conditionally and specifically produced Mafa in db/db islet ß-cells. Sustained expression of Mafa resulted in significantly lower plasma glucose levels, higher plasma insulin, and augmented islet ß-cell mass. In addition, there was increased expression of insulin, Slc2a2, and newly identified Mafa-regulated genes involved in reducing ß-cell stress, like Gsta1 and Gckr. Importantly, the levels of human GSTA1 were also compromised in T2DM islets. Collectively, these results illustrate how consequential the reduction in Mafa activity is to islet ß-cell function under pathophysiological conditions.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Islotes Pancreáticos/metabolismo , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The transcription factor Pdx1 is crucial to islet ß cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using ß cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in ß cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in ß cells (Set(Δ)ß). Set(Δ)ß mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and ß cell function.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Lisina/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Homeodominio/genética , Humanos , Immunoblotting , Lisina/genética , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Células 3T3 NIH , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Transactivadores/genética , Transcripción GenéticaRESUMEN
Analysis of MafB(-/-) mice has suggested that the MAFB transcription factor was essential to islet α- and ß-cell formation during development, although the postnatal physiological impact could not be studied here because these mutants died due to problems in neural development. Pancreas-wide mutant mice were generated to compare the postnatal significance of MafB (MafB(Δpanc)) and MafA/B (MafAB(Δpanc)) with deficiencies associated with the related ß-cell-enriched MafA mutant (MafA(Δpanc)). Insulin(+) cell production and ß-cell activity were merely delayed in MafB(Δpanc) islets until MafA was comprehensively expressed in this cell population. We propose that MafA compensates for the absence of MafB in MafB(Δpanc) mice, which is supported by the death of MafAB(Δpanc) mice soon after birth from hyperglycemia. However, glucose-induced glucagon secretion was compromised in adult MafB(Δpanc) islet α-cells. Based upon these results, we conclude that MafB is only essential to islet α-cell activity and not ß-cell. Interestingly, a notable difference between mice and humans is that MAFB is coexpressed with MAFA in adult human islet ß-cells. Here, we show that nonhuman primate (NHP) islet α- and ß-cells also produce MAFB, implying that MAFB represents a unique signature and likely important regulator of the primate islet ß-cell.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/fisiología , Factor de Transcripción MafB/fisiología , Adolescente , Adulto , Animales , Biomarcadores/metabolismo , Femenino , Humanos , Macaca mulatta , Factor de Transcripción MafB/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Primates , Roedores , Adulto JovenRESUMEN
BACKGROUND: The development of metastases is a negative prognostic parameter for the clinical outcome of breast cancer. Bone constitutes the first site of distant metastases for many affected women. The purpose of this retrospective multicentre study was to evaluate if and how different variables such as primary tumour stage, biological and histological subtype, age at primary diagnosis, tumour size, the number of affected lymph nodes as well as grading influence the development of bone-only metastases. METHODS: This retrospective German multicentre study is based on the BRENDA collective and included 9625 patients with primary breast cancer recruited from 1992 to 2008. In this analysis, we investigated a subgroup of 226 patients with bone-only metastases. Association between bone-only relapse and clinico-pathological risk factors was assessed in multivariate models using the tree-building algorithms "exhausted CHAID (Chi-square Automatic Interaction Detectors)" and CART(Classification and Regression Tree), as well as radial basis function networks (RBF-net), feedforward multilayer perceptron networks (MLP) and logistic regression. RESULTS: Multivariate analysis demonstrated that breast cancer subtypes have the strongest influence on the development of bone-only metastases (χ2 = 28). 29.9 % of patients with luminal A or luminal B (ABC-patients) and 11.4 % with triple negative BC (TNBC) or HER2-overexpressing tumours had bone-only metastases (p < 0.001). Five different mathematical models confirmed this correlation. The second important risk factor is the age at primary diagnosis. Moreover, BC subcategories influence the overall survival from date of metastatic disease of patients with bone-only metastases. Patients with bone-only metastases and TNBC (p < 0.001; HR = 7.47 (95 % CI: 3.52-15.87) or HER2 overexpressing BC (p = 0.007; HR = 3.04 (95 % CI: 1.36-6.80) have the worst outcome compared to patients with luminal A or luminal B tumours and bone-only metastases. CONCLUSION: The bottom line of different mathematical models is the prior importance of subcategories of breast cancer and the age at primary diagnosis for the appearance of osseous metastases. The primary tumour stage, histological subtype, tumour size, the number of affected lymph nodes, grading and NPI seem to have only a minor influence on the development of bone-only metastases.
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Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Ganglios Linfáticos/patología , Recurrencia Local de Neoplasia/patología , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Óseas/metabolismo , Neoplasias de la Mama/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Ganglios Linfáticos/metabolismo , Metástasis Linfática , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Recurrencia Local de Neoplasia/metabolismo , Estadificación de Neoplasias , Pronóstico , Receptor ErbB-2/metabolismo , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Mammography and ultrasound are the gold standard imaging techniques for preoperative assessment and for monitoring the efficacy of neoadjuvant chemotherapy in breast cancer. Maximum accuracy in predicting pathological tumor size non-invasively is critical for individualized therapy and surgical planning. We therefore aimed to assess the accuracy of tumor size measurement by ultrasound and mammography in a multicentered health services research study. METHODS: We retrospectively analyzed data from 6543 patients with unifocal, unilateral primary breast cancer. The maximum tumor diameter was measured by ultrasound and/or mammographic imaging. All measurements were compared to final tumor diameter determined by postoperative histopathological examination. We compared the precision of each imaging method across different patient subgroups as well as the method-specific accuracy in each patient subgroup. RESULTS: Overall, the correlation with histology was 0.61 for mammography and 0.60 for ultrasound. Both correlations were higher in pT2 cancers than in pT1 and pT3. Ultrasound as well as mammography revealed a significantly higher correlation with histology in invasive ductal compared to lobular cancers (p < 0.01). For invasive lobular cancers, the mammography showed better correlation with histology than ultrasound (p = 0.01), whereas there was no such advantage for invasive ductal cancers. Ultrasound was significantly superior for HR negative cancers (p < 0.001). HER2/neu positive cancers were also more precisely assessed by ultrasound (p < 0.001). The size of HER2/neu negative cancers could be more accurately predicted by mammography (p < 0.001). CONCLUSION: This multicentered health services research approach demonstrates that predicting tumor size by mammography and ultrasound provides accurate results. Biological tumor features do, however, affect the diagnostic precision.
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Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/diagnóstico por imagen , Carcinoma Lobular/patología , Anciano , Mama/patología , Mama/cirugía , Neoplasias de la Mama/cirugía , Carcinoma Ductal de Mama/cirugía , Carcinoma Lobular/cirugía , Femenino , Humanos , Mamografía , Persona de Mediana Edad , Estadificación de Neoplasias , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Estudios Retrospectivos , Ultrasonografía MamariaRESUMEN
AIMS/HYPOTHESIS: Several forkhead box (FOX) transcription factor family members have important roles in controlling pancreatic cell fates and maintaining beta cell mass and function, including FOXA1, FOXA2 and FOXM1. In this study we have examined the importance of FOXP1, FOXP2 and FOXP4 of the FOXP subfamily in islet cell development and function. METHODS: Mice harbouring floxed alleles for Foxp1, Foxp2 and Foxp4 were crossed with pan-endocrine Pax6-Cre transgenic mice to generate single and compound Foxp mutant mice. Mice were monitored for changes in glucose tolerance by IPGTT, serum insulin and glucagon levels by radioimmunoassay, and endocrine cell development and proliferation by immunohistochemistry. Gene expression and glucose-stimulated hormone secretion experiments were performed with isolated islets. RESULTS: Only the triple-compound Foxp1/2/4 conditional knockout (cKO) mutant had an overt islet phenotype, manifested physiologically by hypoglycaemia and hypoglucagonaemia. This resulted from the reduction in glucagon-secreting alpha cell mass and function. The proliferation of alpha cells was profoundly reduced in Foxp1/2/4 cKO islets through the effects on mediators of replication (i.e. decreased Ccna2, Ccnb1 and Ccnd2 activators, and increased Cdkn1a inhibitor). Adult islet Foxp1/2/4 cKO beta cells secrete insulin normally while the remaining alpha cells have impaired glucagon secretion. CONCLUSIONS/INTERPRETATION: Collectively, these findings reveal an important role for the FOXP1, 2, and 4 proteins in governing postnatal alpha cell expansion and function.
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Proliferación Celular , Factores de Transcripción Forkhead/metabolismo , Células Secretoras de Glucagón/metabolismo , Proteínas Represoras/metabolismo , Animales , Factores de Transcripción Forkhead/genética , Glucagón/sangre , Células Secretoras de Glucagón/citología , Insulina/sangre , Ratones , Ratones Transgénicos , Proteínas Represoras/genéticaRESUMEN
An appropriate beta cell mass is pivotal for the maintenance of glucose homeostasis. Both insulin and IGF-1 are important in regulation of beta cell growth and function (reviewed in ref. 2). To define the roles of these hormones directly, we created a mouse model lacking functional receptors for both insulin and IGF-1 only in beta cells (betaDKO), as the hormones have overlapping mechanisms of action and activate common downstream proteins. Notably, betaDKO mice were born with a normal complement of islet cells, but 3 weeks after birth, they developed diabetes, in contrast to mild phenotypes observed in single mutants. Normoglycemic 2-week-old betaDKO mice manifest reduced beta cell mass, reduced expression of phosphorylated Akt and the transcription factor MafA, increased apoptosis in islets and severely compromised beta cell function. Analyses of compound knockouts showed a dominant role for insulin signaling in regulating beta cell mass. Together, these data provide compelling genetic evidence that insulin and IGF-I-dependent pathways are not critical for development of beta cells but that a loss of action of these hormones in beta cells leads to diabetes. We propose that therapeutic improvement of insulin and IGF-I signaling in beta cells might protect against type 2 diabetes.
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Diabetes Mellitus Experimental/fisiopatología , Factor I del Crecimiento Similar a la Insulina/fisiología , Insulina/fisiología , Islotes Pancreáticos/fisiopatología , Animales , Diabetes Mellitus Experimental/etiología , Humanos , Espectrometría de Masas , Ratones , Ratones Noqueados , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/fisiología , Receptor de Insulina/genética , Receptor de Insulina/fisiologíaRESUMEN
MafA and Pdx1 represent critical transcriptional regulators required for the maintenance of pancreatic islet ß-cell function. The in vivo ß-cell-enriched expression pattern of these genes is principally directed by islet transcription factors binding within conserved Region 3 (base pairs (bp) -8118/-7750) of MafA and Area II (bp -2153/-1923) of the Pdx1 gene. Comprehensive mutational analysis of conserved MafA Region 3 revealed two new ß-cell line-specific cis-activation elements, termed Site 4 (bp -7997 to -7988) and Site 12 (bp -7835 to -7826). Gel mobility and antibody super-shift analysis identified Pdx1 as the Site 4 binding factor, while an 80-88 kilodalton (kDa) ß-cell line-enriched protein complex bound Site 12 and similar aligned nucleotides within Pdx1 Area II. The 80-88 kDa activator was also found in adult mouse islet extract. Strikingly, the molecular weight, DNA binding, and antibody recognition properties of this activator were unique when compared with all other key islet transcription factors tested, including Prox1 (83 kDa), Hnf1α (67 kDa), FoxA2 (48 kDa), MafA (46 kDa), Isl1 (44 kDa), Pdx1 (42 kDa), and Nkx2.2 (30 kDa). Collectively, these data define an apparently novel MafA Region 3 and Pdx1 Area II activator contributing to expression in ß-cells.
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Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Elementos de Respuesta/fisiología , Transactivadores/metabolismo , Animales , Línea Celular , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/citología , Factores de Transcripción Maf de Gran Tamaño/genética , Ratones , Transactivadores/genéticaRESUMEN
BACKGROUND: Gestational diabetes mellitus (GDM) occurs in 3-5% of all pregnancies. GDM increases both maternal and fetal risks, causes fetal macrosomia, and hence increases the rates of caesarean sections and delivery complications such as shoulder dystocia. An early predictive marker and consequent early treatment could be beneficial, so amniotic fluid insulin and C-peptide have been examined in several studies. Increased amniotic fluid insulin in early amniocentesis between the 14th and 20th gestational week predicted a later GDM. A potential direct association with fetal macrosomia remains to be determined. MATERIAL AND METHODS: This retrospective study investigated amniotic fluid insulin/C-peptide from amniocenteses between 14 and 20 weeks of gestation in correlation with fetal birth weight, type of delivery, and complications. To focus on effects of fetal hyperinsulinism apart from therapeutic confounders, we included patients who did not participate in GDM screening. Insulin and C-peptide were measured in 144 samples of frozen amniotic fluid. Birth weight, type of delivery, complications, and birth injuries were noted. RESULTS: Birth weights ranged from 760 g to 4410 g with a mean weight of 3424 g at an average of 40 weeks gestation. The mean amniotic fluid insulin was 4.36 U/ml and the mean C-peptide concentration was 0.076 ng/ml. There was no correlation between amniotic fluid insulin or C peptide and birth weight, type of delivery, complications, and birth injuries. CONCLUSIONS: Amniotic fluid insulin and C-peptide are unsuitable as predictive marker for fetal macrosomia, type of delivery, complications, or birth injuries.
Asunto(s)
Biomarcadores/metabolismo , Traumatismos del Nacimiento/diagnóstico , Complicaciones de la Diabetes/diagnóstico , Diabetes Gestacional/diagnóstico , Macrosomía Fetal/diagnóstico , Complicaciones del Trabajo de Parto/diagnóstico , Amniocentesis , Líquido Amniótico/química , Biomarcadores/análisis , Traumatismos del Nacimiento/etiología , Traumatismos del Nacimiento/metabolismo , Peso al Nacer , Péptido C/análisis , Complicaciones de la Diabetes/metabolismo , Diabetes Gestacional/metabolismo , Femenino , Macrosomía Fetal/etiología , Macrosomía Fetal/metabolismo , Humanos , Insulina/análisis , Radioisótopos de Yodo/análisis , Complicaciones del Trabajo de Parto/etiología , Complicaciones del Trabajo de Parto/metabolismo , Embarazo , Estudios RetrospectivosRESUMEN
A hallmark of type 2 diabetes (T2D) is endocrine islet ß-cell failure, which can occur via cell dysfunction, loss-of-identity, and/or death. How each is induced remains largely unknown. Here, we use mouse ß-cells that are deficient for Myelin transcription factors (Myt TFs, including Myt1, 2, and 3) to address this question. We have reported that inactivating all three Myt genes in pancreatic progenitor cells (MytPancΔ) causes ß-cell failure and late onset diabetes in mice. Their lower expression in human ß-cells is correlated with ß-cell dysfunction and SNPs in MYT2 and MYT3 are associated in higher risk of T2D. We now show that these Myt TF-deficient postnatal ß-cells also de-differentiate by reactivating several progenitor markers. Intriguingly, mosaic Myt TF inactivation in only a portion of islet ß-cells does not results in overt diabetes, but this creates a condition where Myt TF-deficient ß-cells stay alive while activating several markers of Ppy-expressing islet cells. By transplanting MytPancΔ islets into the anterior eye chambers of immune-compromised mice, we directly show that glycemic and obesity-related conditions influence cell fate, with euglycemia inducing several Ppy+ cell markers while hyperglycemia and insulin resistance inducing additional cell death. These findings suggest that the observed ß-cell defects in T2D depend on not only their inherent genetic/epigenetic defects, but also the metabolic load.
RESUMEN
Interactions between lineage-determining and activity-dependent transcription factors determine single-cell identity and function within multicellular tissues through incompletely known mechanisms. By assembling a single-cell atlas of chromatin state within human islets, we identified ß cell subtypes governed by either high or low activity of the lineage-determining factor pancreatic duodenal homeobox-1 (PDX1). ß cells with reduced PDX1 activity displayed increased chromatin accessibility at latent nuclear factor κB (NF-κB) enhancers. Pdx1 hypomorphic mice exhibited de-repression of NF-κB and impaired glucose tolerance at night. Three-dimensional analyses in tandem with chromatin immunoprecipitation (ChIP) sequencing revealed that PDX1 silences NF-κB at circadian and inflammatory enhancers through long-range chromatin contacts involving SIN3A. Conversely, Bmal1 ablation in ß cells disrupted genome-wide PDX1 and NF-κB DNA binding. Finally, antagonizing the interleukin (IL)-1ß receptor, an NF-κB target, improved insulin secretion in Pdx1 hypomorphic islets. Our studies reveal functional subtypes of single ß cells defined by a gradient in PDX1 activity and identify NF-κB as a target for insulinotropic therapy.
Asunto(s)
Células Secretoras de Insulina , FN-kappa B , Animales , Humanos , Ratones , Cromatina/metabolismo , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , FN-kappa B/metabolismoRESUMEN
Glucolipotoxicity, caused by combined hyperglycemia and hyperlipidemia, results in ß-cell failure and type 2 diabetes (T2D) via cellular stress-related mechanisms. Activating transcription factor 4 (Atf4) is an essential effector of stress response. We show here that Atf4 expression in ß-cells is dispensable for glucose homeostasis in young mice, but it is required for ß-cell function during aging and under obesity-related metabolic stress. Henceforth, aged Atf4- deficient ß-cells display compromised secretory function under acute hyperglycemia. In contrast, they are resistant to acute free fatty acid-induced loss-of identity and dysfunction. At molecular level, Atf4 -deficient ß-cells down-regulate genes involved in protein translation, reducing ß-cell identity gene products under high glucose. They also upregulate several genes involved in lipid metabolism or signaling, likely contributing to their resistance to free fatty acid-induced dysfunction. These results suggest that Atf4 activation is required for ß-cell identity and function under high glucose, but this paradoxically induces ß-cell failure in the presence of high levels of free fatty acids. Different branches of Atf4 activity could be manipulated for protecting ß-cells from metabolic stress-induced failure. Highlights: Atf4 is dispensable in ß-cells in young miceAtf4 protects ß-cells under high glucoseAtf4 exacerbate fatty acid-induced ß-cell defectsAtf4 activates translation but depresses lipid-metabolism.
RESUMEN
The FOXO1 (forkhead box O1) transcription factor influences many key cellular processes, including those important in metabolism, proliferation and cell death. Reversible phosphorylation of FOXO1 at Thr(24) and Ser(256) regulates its subcellular localization, with phosphorylation promoting cytoplasmic localization, whereas dephosphorylation triggers nuclear import and transcriptional activation. In the present study, we used biochemical and molecular approaches to isolate and link the serine/threonine PP2A (protein phosphatase 2A) holoenzyme containing the B55α regulatory subunit, with nuclear import of FOXO1 in pancreatic islet ß-cells under oxidative stress, a condition associated with cellular dysfunction in Type 2 diabetes. The mechanism of FOXO1 dephosphorylation and nuclear translocation was investigated in pancreatic islet INS-1 and ßTC-3 cell lines subjected to oxidative stress. A combined chemical cross-linking and MS strategy revealed the association of FOXO1 with a PP2A holoenzyme composed of the catalytic C, structural A and B55α regulatory subunits. Knockdown of B55α in INS-1 cells reduced FOXO1 dephosphorylation, inhibited FOXO1 nuclear translocation and attenuated oxidative stress-induced cell death. Furthermore, both B55α and nuclear FOXO1 levels were increased under hyperglycaemic conditions in db/db mouse islets, an animal model of type 2 diabetes. We conclude that B55α-containing PP2A is a key regulator of FOXO1 activity in vivo.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Células Secretoras de Insulina/metabolismo , Estrés Oxidativo/fisiología , Proteína Fosfatasa 2/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Proteína Forkhead Box O1 , Células HEK293 , Humanos , Células Secretoras de Insulina/enzimología , Islotes Pancreáticos/metabolismo , Isoenzimas/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fosforilación/fisiología , Ratas , Regulación hacia Arriba/fisiologíaRESUMEN
MAFA and MAFB are related basic-leucine-zipper domain containing transcription factors which have important regulatory roles in a variety of cellular contexts, including pancreatic islet hormone producing α and ß cells. These proteins have similar as well as distinct functional properties, and here we first used AlphaFold2, an artificial intelligence-based structural prediction program, to obtain insight into the three-dimensional organization of their non-DNA binding/dimerization sequences. This analysis was conducted on the wildtype (WT) proteins as well the pathogenic MAFA Ser64Phe (MAFA S64F ) and MAFB Ser70Ala (MAFB S70A ) mutants, with structural differences revealed between MAFA WT and MAFB WT in addition to MAFA S64F and MAFA WT , but not MAFB S70A and MAFB WT . Functional analysis disclosed that the inability to properly phosphorylate at S70 in MAFB S70A , like S65 in MAFA S64F , greatly increased protein stability and enabled MAFB S70A to accelerate cellular senescence in cultured cells. Significant differences were also observed in the ability of MAFA, MAFA S64F , MAFB, and MAFB S70A to cooperatively stimulate Insulin enhancer-driven activity in the presence of other islet-enriched transcription factors. Experiments performed on protein chimeras disclosed that these properties were greatly influenced by structural differences found between the WT and mutant proteins. In general, these results revealed that AlphaFold2 predicts features essential to protein activity.